Specific Aim 1. Activity-dependent regulation of axonal mitochondrial transport Axonal mitochondria are recruited to synaptic terminals in response to neuronal activity. However, the mechanisms underlying activity-dependent regulation of mitochondrial transport are largely unknown. Recent advances in identifying SNPH as a mitochondrial docking protein and KIF5 motor adaptor Miro as a calcium sensor in arresting mitochondrial movement provide molecular targets for such regulation. Using genetic models combined with live imaging, we recently demonstrated that syntaphilin (SNPH) mediates the activity-dependent immobilization of axonal mitochondria through binding to KIF5 motors. In vitro analysis showed that the KIF5-SNPH coupling inhibits the motor ATPase. Neuronal activity further recruits SNPH to axonal mitochondria. This motor-docking interplay is induced by Ca2+ and synaptic activity and is necessary to establish an appropriate balance between motile and stationary axonal mitochondria. Deleting snph abolishes the activity-dependent immobilization of axonal mitochondria. We propose an Engine-Switch and Brake model, in which SNPH acts both as an engine off-switch by sensing Miro-Ca2+ and as a brake by anchoring mitochondria to the MT-track. Altogether, our study provides new mechanistic insight into the molecular interplay between motor and docking proteins, which arrests axonal mitochondrial transport in response to changes in neuronal activity. Our Engine-Switch and Brake model nicely reconciles the current dispute in explaining how Miro-Ca2+ sensing mediates the suppression of mitochondrial transport in axons and dendrites (MacAskill et al., 2009b; Wang and Schwarz, 2009). Thus, our study elucidates a new molecular mechanism underlying the complex regulation of axonal mitochondrial transport, thereby advancing our knowledge that may be essential for maintaining axonal and synaptic homeostasis (Chen and Sheng, Journal of Cell Biology 2013). Specific Aim 2. Mobile axonal mitochondria contribute to the variability of synaptic strength One of the most notable characteristics of synaptic physiology in the CNS is the wide pulse-to-pulse variability of synaptic strength in response to identical stimulation. A long-standing question is how this variability arises. In hippocampal neurons, approximately one-third of mitochondria are highly motile and some dynamically pass through presynaptic boutons. This raises a fundamental question: Can those motile mitochondria contribute to the pulse-to-pulse variability of presynaptic strength? Deleting the snph gene recruited the majority of axonal mitochondria into motile pools while over-expressing SNPH abolished axonal mitochondrial motility. Thus, the snph mouse provides us with a unique genetic tool to experimentally address whether selective changes in axonal mitochondrial motility could compromise the variability of synaptic strength. Our recent study has solved this puzzle by combining live neuron dual-color imaging and electrophysiological analysis of genetic mouse model in which axonal mitochondrial motility was selectively manipulated. Using hippocampal neurons and slices of snph knockout mice, we demonstrate that the motility of axonal mitochondria correlates with presynaptic variability. Enhancing mitochondrial motility increases the pulse-to-pulse variability, while immobilizing mitochondria reduces the variability. By live imaging at the single-bouton level, we further show that motile mitochondria passing through boutons dynamically influence synaptic vesicle release, mainly through altered ATP homeostasis in axons. The absence of mitochondria within presynaptic terminals reduces local ATP supply, thus impairing ATP-dependent processes including SV pool replenishment. Although presynaptic mitochondria are crucial to maintain the proper size of functional releasable SV pools during repeated trains of stimulation, it is possible that other ATP-dependent processes may collectively contribute to synaptic variability when mitochondria dynamically travel along axons and move into or pass by boutons. The effects of synaptic variability on neuronal or circuit activity as well as information coding and processing are increasingly recognized. Thus, our study brings new insight into the fundamental properties of the CNS to ensure the plasticity and reliability of synaptic transmission. Our study revealed, for the first time, that the dynamic movement of axonal mitochondria is one of the primary mechanisms underlying the pulse-to-pulse variability of presynaptic strength and the trial-to-trial variation in the efficiency of SV release in the CNS (Sun et al., Cell Reports 2013). Specific Aim 3. Mitochondrial mobility regulates their quality control via the Parkin-mediated mitophagy PINK1/Parkin-mediated pathways ensure mitochondrial integrity and function. Parkin translocation to damaged mitochondria induces mitophagy in many non-neuronal cell types. However, evidence showing Parkin-mediated mitophagy in primary neurons is controversial. Our recent study reveals several unique features of Parkin-mediated mitophagy and subsequent degradation of dysfunctional mitochondria in live mature cortical neurons (Cai et al., Current Biology 2012; Cai et al., Autophagy 2012). (1) Parkin translocation onto depolarized mitochondria is a much slower process than in non-neuronal cells and occurs in a small portion (30 %) of neurons following CCCP treatment for 24 hrs. (2) Depolarized mitochondria in axons first undergo relatively enhanced retrograde transport to soma where Parkin translocation then occurs. (3) As consequence, Parkin-targeted mitochondria are restricted to the somatodendritic regions, where mature lysosomes are predominantly localized. (4) Anterograde transport of dysfunctional mitochondria was reduced, thus preventing them from traveling peripherally. Therefore, altered mitochondrial mobility may be protective for neurons under stressful conditions, where healthy mitochondria remain distally while damaged mitochondria return to the soma for degradation. Specific Aim 4. Impact of mitochondrial motility on axonal degeneration. Mitochondrial dysfunction and defective axonal transport have been implicated in the pathogenesis of major neurodegenerative diseases. However, whether altered mitochondrial transport alone plays a critical role in axonal degeneration has been a subject for debate. Our snph mouse provides a valuable model to assess the direct impact of mitochondrial mobility on axonal degeneration. Amyotrophic lateral sclerosis (ALS) is a late onset neurodegenerative disease specifically affecting motor neurons. We selected ALS-linked SOD1G93A mutant mice for our study. By crossing SOD1G93A and snph-/- mice we addressed whether increasing (rescued) mitochondrial mobility has any impact on the pathogenesis of the fALS-linked SOD1G93A mouse model. The crossed mice exhibit a 2-fold increase in axonal mitochondrial mobility at late disease stages. To our surprise, there is no observable improvement in the deterioration of motor function or in disease progression and lifespan. The crossed mice show similar ALS-like disease histopathology that includes motor neuron loss and gliosis. Our study suggests that elevating mitochondrial transport alone is not sufficient to slow or reverse mitochondrial dysfunction and motor neuron degeneration. Thus, a combined approach by rescuing mitochondrial transport and enhancing mitophagy-lysosomal system is suggested in our future study to ensure mitochondrial integrity and function in rapid-onset motor neuron degeneration (Zhu and Sheng JBC, 2011). In summery, continued pursuing these investigations will advance our knowledge of fundamental processes that may affect human neurological disorders and is thus the very essence of the mission of NINDS, NIH.